Inhibition of NF-kappaB signalling by a family of type III secretion system effector proteins during Salmonella infection

During Salmonella infection, bacterial proteins called ‘effectors’ are translocated into host cells by two type III secretion system apparatuses encoded by Salmonella-pathogenicity island 1 and 2. These effectors manipulate host cell processes to facilitate the formation of an intracellular replicative niche, to prevent bacterial clearance, and ultimately promote bacterial transmission to another susceptible host. A subset of SPI-2 T3SS effector proteins manipulate innate immune signalling pathways thereby preventing formation of an appropriate immune response. In this thesis, I identify three related effector proteins - GtgA, GogA, and PipA, as sufficient to inhibit NF-kappaB signalling when expressed ectopically. Furthermore, I demonstrate that GtgA, GogA, and PipA are zinc metalloproteases that inhibit NF-kappaB signalling by cleaving the NF-kappaB transcription factor subunits p65, cRel, and RelB, but not NF-kappaB1 (p105/p50) or NF-kappaB2 (p100/p52). Accordingly, in Salmonella-infected cells, p65 was cleaved dependent on gtgA, gogA, and pipA leading to inhibition of NF-kappaB signalling. To investigate the molecular basis for substrate recognition, mutational analysis of residues in close proximity to the p65 cleavage site (G40/R41) was done and showed that the P1’ residue (R41 in p65) is a critical determinant of substrate specificity. In NF-kappaB1 and NF-kappaB2, a proline residue is present at the corresponding site and this residue prevents cleavage by GtgA, GogA, and PipA. I also present the crystal structure of GtgA alone and in complex with the N-terminal domain of p65. The crystal structure demonstrates the importance of the P1’ residue in substrate specificity and supports a model of DNA mimicry as the mechanism of substrate recognition. This thesis therefore provides detailed insight into the functions and mechanism of substrate recognition, for a family of previously uncharacterised Salmonella virulence proteins.Open Acces